PROJECT 2 SUMMARY (CRISPR/Cas9 and small molecules for targeting sperm function and fertilization) The overall goals of Project 2 are to use CRISPR/Cas9 to understand the formation of functional sperm and identify small-molecule probes and preclinical candidates to target fertilization-required proteins for a contraceptive effect in vivo. Population growth is a major worldwide issue, and our resources cannot continue to sustain these population increases, and because of this, NICHD has made contraception a priority focus area. Because our group is focused on the development of contraceptives that specifically target sperm and fertilization, these germ cell-selective contraceptives would eliminate unwanted side effects. The Ikawa laboratory has developed the CRISPR/Cas9 system to efficiently mutate genes in vivo including the Cetn1 and Prm1 genes required for male fertility. In this P01, we will use CRISPR/Cas9 technology to advance our understanding of fertility pathways in all three Projects and generate useful information to move these target proteins and other related pathway proteins into the DEC-Tec Core for screening. For contraceptive targets in Project 2, we will focus on two sperm-egg fusion transmembrane/signaling proteins and four sperm motility- related proteins that we have shown are evolutionarily-conserved and required only for male fertility. The Ikawa laboratory has effectively used the CRISPR/Cas9 system to generate mutations in over 200 mouse genes including the genes described in this proposal. This strategy has also successfully produced knockin of tag sequences into loci. We have used CRISPR/Cas9 independently and in collaboration with the Matzuk laboratory (Project 1) to uncover proteins required for sperm-egg fusion and sperm motility. Functional analysis of these mouse models will help not only to decipher the mechanisms by which these proteins function in sperm-egg fusion and sperm motility but also to develop contraceptive drugs to target these essential pathways. The screening of small molecules using the DEC-TEC Core will accelerate this process. Our overall hypothesis is that CRISPR/Cas9 and DEC-Tec will help us to understand the network of interactions of these proteins for sperm function, rapidly identify multiple small molecules that are directed at these essential spermatogenic proteins, and create an assortment of contraceptives for men and women. The Specific Aims of Project 2 are: 1) Manipulate the mouse genome and characterize the interrelationship of sperm-egg fusion proteins; 2) Use CRISPR/Cas9 to study the mechanism of action of novel testis-specific proteins; and 3) Use DEC-Tec to identify small-molecule drug-like probes and preclinical candidates to inhibit sperm motility-specific and fertilization-specific proteins for a contraceptive effect in vivo. The success of Project 2 and this P01 grant relies on the continued collaborations of Project 2 with Dr. Matzuk in Project 1, Drs. Sonnenburg, Lamb, and Huang in Project 3, and the scientists in the DEC-Tec Core. Our goal is to develop multiple unique transgenic models and non-hormonal contraceptives for mechanistic and pharmacologic studies in vivo in this P01 grant.